1. Field of the Invention
The present invention relates generally to biologically important phosphates such as second messengers. More particularly, the present invention relates to modifying second messengers to form derivatives which can be introduced into a cell without disrupting the cell membrane. Once inside the cell, the derivative is converted back to the biologically active second messenger.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically reference and grouped in the appended bibliography.
Second messengers are ions or small molecules that carry information from the cell membrane to targets on the inside of the cell. They play a major role in biological signal transduction and amplification (1). A common feature of most of the known second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) (2,3), guanosine 3',5'-cyclic monophosphate (4) (cGMP), myo-inositol-1,4,5-triphosphate (1,4,5)IP.sub.3) or myo-inositol-1,3,4,5-tetrakisphosphate (1,3,4,5)IP.sub.4) (5), is the presence of phosphates. The correct number and position of these phosphates is essential for biological specificity and also confers extreme hydrophilicity (6,7).
The hydrophilicity of second messengers prevents endogenously generated molecules from leaking out of cells. As a result, high sensitivity is maintained within the responding cell and freedom from cross-talk between neighboring cells is achieved. However, the membrane impermeability of second messengers also makes deliberate extracellular application difficult or ineffective (2,6,8), even though such intervention would often be very useful for research or therapeutic reasons.
One approach for introducing phosphate containing compounds into cells involves using protective groups to reversibly convert the negatively charged phosphate compound into a neutral compound for transport through the cell membrane. The protective group is chosen so that it is enzymatically cleaved from the phosphate compound inside the cell to produce the original phosphate compound. For example, lipophilic, intracellularly hydrolyzable derivatives have been useful for amino, hydroxyl, and carboxylate moieties (9-12). Acetoxymethyl (AM) esters of polycarboxylate cation indicators and chelators are have also been used (12-14). Analogous acyloxyalkyl esters applied to phosphates are known, but they have been less widely exploited (15).
On simple model phosphates, uses of AM esters have been limited to potential therapeutic drugs, such as phosphonoformate (foscarnet) (16), antiviral nucleotides such as 5-fluoro-2'-deoxyuridine monophosphate (17,18), and a 3-phosphonate-containing inhibitor of the insulin receptor kinase (19). The phosphonoformate esters proved not to be biologically useful due to failure to hydrolyze to the correct products (16), but esterification was found to enhance the effectiveness of the antiviral nucleotides and kinase inhibitor (17-19).
Considerable work has been done on o-nitrobenzyl esters as photolyzable ("caged") derivatives of ATP (20), cyclic nucleotides (21,22), and inositol phosphates (23). However, the emphasis has been on producing a kinetically fast and complete transition from a monoester to the active freed phosphate metabolite (24,25), rather than as a general means of achieving membrane permeability. In addition, nitrobenzyl esters become cumbersome if more than one are required to mask negative charges, because multiple groups add considerable bulk and require high doses of UV to cause cleavage of all the groups.
Although numerous different myo-inositol polyphosphates are possible, only about a dozen have been found in cells. Their intracellular functions are controversial or unknown, except for myo-inositol 1,4,5-triphosphate (IP.sub.3), whose role as an intracellular second messenger to release Ca.sup.2+ from internal stores is unquestioned (26). The next most studied inositol polyphosphate is myo-inositol 1,3,4,5-tetrakisphosphate (IP.sub.4), which is believed to cooperate with IP.sub.3 to open Ca.sup.2+ -channels in the plasma membrane (27-30) or to resequester Ca.sup.2+ released by IP.sub.3 (31, 32). However, these hypotheses remain controversial (33-37).
Almost nothing is known about intracellular functions for other inositol polyphosphates. Detailed dissection of the roles of inositol polyphosphates is often difficult if they are endogenously generated in response to agonists, since such stimulation may affect multiple receptors, G proteins, diacylglycerol formation, multiple inositol polyphosphates, and yet other transduction pathways. Direct introduction of specific inositol polyphosphates is often preferable.
The high negative charge of inositol polyphosphates results in negligible passive permeability through membranes. Existing methods for introducing inositol phosphates include microinjection, patch-clamp techniques, and permeabilization by electroporation, detergents like saponin, or removal of extracellular Ca.sup.2+. All these methods breach the plasma membrane and jeopardize the more complex functions and long-term viability of the cells. Furthermore, microinjection and patch techniques can only be applied to a few cells at a time.
In view of the above, there is a need to provide new compounds and procedures for increasing the membrane permeability of second messengers so that they can be introduced effectively into cells in amounts which are useful for investigational or therapeutic purposes.